Description: Objective Acute pancreatitis is caused by toxins that induce acinar cell calcium overload, zymogen activation, cytokine release and cell death, yet is without specific drug therapy. Mitochondrial dysfunction has been implicated but the mechanism not established. Design We investigated the mechanism of induction and consequences of the mitochondrial permeability transition pore (MPTP) in the pancreas using cell biological methods including confocal microscopy, patch clamp technology and multiple clinically representative disease models. Effects of genetic and pharmacological inhibition of the MPTP were examined in isolated murine and human pancreatic acinar cells, and in hyperstimulation, bile acid, alcoholic and choline-deficient, ethionine-supplemented acute pancreatitis. Results MPTP opening was mediated by toxin-induced inositol trisphosphate and ryanodine receptor calcium channel release, and resulted in diminished ATP production, leading to impaired calcium clearance, defective autophagy, zymogen activation, cytokine production, phosphoglycerate mutase 5 activation and necrosis, which was prevented by intracellular ATP supplementation. When MPTP opening was inhibited genetically or pharmacologically, all biochemical, immunological and histopathological responses of acute pancreatitis in all four models were reduced or abolished. Conclusions This work demonstrates the mechanism and consequences of MPTP opening to be fundamental to multiple forms of acute pancreatitis and validates the MPTP as a drug target for this disease.

Description: Objective Precancerous lesion, a well-established histopathologically premalignant tissue with the highest risk for tumourigenesis, develops preferentially from activation of DNA damage checkpoint and persistent inflammation. However, little is known about the mechanisms by which precancerous lesions are initiated and their physiological significance. Design Laser capture microdissection was used to acquire matched normal liver, precancerous lesion and tumour tissues. miR-484 –/– , Ifnar1 –/– and Tgfbr2 hep mice were employed to determine the critical role of the interferon (IFN)–microRNA pathway in precancerous lesion formation and tumourigenesis. RNA immunoprecipitation (RIP), pull-down and chromatin immunoprecipitation (ChIP) assays were applied to explore the underlying mechanisms. Results miR-484 is highly expressed in over 88% liver samples clinically. DEN-induced precancerous lesions and hepatocellular carcinoma were dramatically impaired in miR-484 –/– mice. Mechanistically, ectopic expression of miR-484 initiates tumourigenesis and cell malignant transformation through synergistic activation of the transforming growth factor-β/Gli and nuclear factor-B/type I IFN pathways . Specific acetylation of H3K27 is indispensable for basal IFN-induced continuous transcription of miR-484 and cell transformation. Convincingly, formation of precancerous lesions were significantly attenuated in both Tgfbr2 hep and Ifnar1 –/– mice. Conclusions These findings demonstrate a new protumourigenic axis involving type I IFN–microRNA signalling, providing a potential therapeutic strategy to manipulate or reverse liver precancerous lesions and tumourigenesis.

Description: Objective Caffeine reduces toxic Ca 2+ signals in pancreatic acinar cells via inhibition of inositol 1,4,5-trisphosphate receptor (IP 3 R)-mediated signalling, but effects of other xanthines have not been evaluated, nor effects of xanthines on experimental acute pancreatitis (AP). We have determined effects of caffeine and its xanthine metabolites on pancreatic acinar IP 3 R-mediated Ca 2+ signalling and experimental AP. Design Isolated pancreatic acinar cells were exposed to secretagogues, uncaged IP 3 or toxins that induce AP and effects of xanthines, non-xanthine phosphodiesterase (PDE) inhibitors and cyclic adenosine monophosphate and cyclic guanosine monophosphate (cAMP/cGMP) determined. The intracellular cytosolic calcium concentration ([Ca 2+ ] C ), mitochondrial depolarisation and necrosis were assessed by confocal microscopy. Effects of xanthines were evaluated in caerulein-induced AP (CER-AP), taurolithocholic acid 3-sulfate-induced AP (TLCS-AP) or palmitoleic acid plus ethanol-induced AP (fatty acid ethyl ester AP (FAEE-AP)). Serum xanthines were measured by liquid chromatography-mass spectrometry. Results Caffeine, dimethylxanthines and non-xanthine PDE inhibitors blocked IP 3 -mediated Ca 2+ oscillations, while monomethylxanthines had little effect. Caffeine and dimethylxanthines inhibited uncaged IP 3 -induced Ca 2+ rises, toxin-induced Ca 2+ release, mitochondrial depolarisation and necrotic cell death pathway activation; cAMP/cGMP did not inhibit toxin-induced Ca 2+ rises. Caffeine significantly ameliorated CER-AP with most effect at 25 mg/kg (seven injections hourly); paraxanthine or theophylline did not. Caffeine at 25 mg/kg significantly ameliorated TLCS-AP and FAEE-AP. Mean total serum levels of dimethylxanthines and trimethylxanthines peaked at 〉2 mM with 25 mg/kg caffeine but at 〈100 µM with 25 mg/kg paraxanthine or theophylline. Conclusions Caffeine and its dimethylxanthine metabolites reduced pathological IP 3 R-mediated pancreatic acinar Ca 2+ signals but only caffeine ameliorated experimental AP. Caffeine is a suitable starting point for medicinal chemistry.